A new method called Multiple Displacement Amplification (MDA) has made it possible to sequence bacterial genomic DNA from a single cell. MDA is used to amplify the few femtograms of DNA in the cell for use in sequencing and for diagnostic and genotyping assays. MDA is rapidly being adopted for investigation of environmental bacterial communities, such as in oceans, where it can be used to discover novel microbes. However, there has been little effort yet to use MDA for studying bacteria living in the human body where powerful new research strategies could be developed to study the normal and pathogenic flora. Our objective is to develop the methods needed for use of MDA in amplifying bacterial genomes from human clinical specimens. Methods will be developed for tissues, fecal samples, body fluids and other specimen types for isolating single cells, lysing them to release their DNA, and carrying out MDA whole genome amplification. Preliminary studies have demonstrated high throughput isolation of Haemophilus influenzae from inner ear mucosa by flow cytometry. Isolation of single cells is also being carried out by micromanipulation in which a glass capillary is used under the microscope and specific bacteria are targeted by labeling with fluorescent DNA probes. Microfluidic MDA on chips is being developed for use in "point of care" diagnostic tests that could be used at any location without lab processing required. Laser capture microdissection (LCM) is being tested as an alternative method of cell isolation for use with infected tissues, particularly where bacterial biofilms or intracellular infection are suspected. Other pathogens to be studied will be Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa from middle ear, and throat tissues and puss from wounds and infected medical implants. Ground braking methods are in development for combining confocal microscopy imaging of tissues and associated bacteria with single cell isolation and genomic sequencing. Bacteria from fecal samples will be used as part of a major project at Washington University in St. Louis to identify the microbes of the human gut. Borrelia burgdorferi, the cause of Lyme disease, has also been isolated from tick gut and DNA sequencing and genotyping demonstrated. Isolation from the characteristic Lyme disease rash in human skin is planned next and ultimately from human brain tissue where it is suspected of causing neurological symptoms. In parallel with the development of these new applications, our work in developing and optimizing the MDA method continues. Research is proposed into the basic enzymology of the MDA reaction and the chemical pathways involved in DNA amplification. Rapid progress is being made to improve the performance of MDA and to perfect methods and strategies for use of amplified DNA by both the Sanger sequencing method and 454 pyrosequencing. The overall goal is to complete the development of methods for in vitro amplification of genomic DNA from singles cells and to demonstrate the research applications of this new technology to the natural human bacterial flora and to infectious disease. [unreadable] [unreadable] [unreadable]